The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the alphabeta T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated alphabeta TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Valpha and Vbeta domains but deviates in the constant Calpha domain and in the interdomain pairing of Calpha with Cbeta. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the Calpha-Cbeta interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarity-determining regions) of the alpha and beta chains. The 2C TCR covers the class I MHC H-2Kb binding groove so that the Valpha CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vbeta chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Valpha and Vbeta CDR3s straddle the peptide between the helices around the central position of the peptide.

Several families of transmembrane endoplasmic reticulum (ER) proteins contain retention motifs in their cytoplasmically exposed tails. Mutational analyses demonstrated that two lysines positioned three and four or five residues from the C‐terminus represent the retention motif. The introduction of a lysine preceding the lysine that occurs three residues from the terminus of Lyt2 renders this cell surface protein a resident of the ER. Likewise, the appropriate positioning of two lysine residues in a poly‐serine sequence confines marker proteins to the ER. Arginines or histidines cannot replace lysines, suggesting that simple charge interactions are not sufficient to explain the retention. The identified consensus motif may serve as a retrieval signal that brings proteins back from a sorting compartment adjacent to the ER.

Histamine regulates neurotransmitter release in the central and peripheral nervous systems through H3 presynaptic receptors. The existence of the histamine H3 receptor was demonstrated pharmacologically 15 years ago, yet despite intensive efforts, its molecular identity has remained elusive. As part of a directed effort to discover novel G protein-coupled receptors through homology searching of expressed sequence tag databases, we identified a partial clone (GPCR97) that had significant homology to biogenic amine receptors. The GPCR97 clone was used to probe a human thalamus library, which resulted in the isolation of a full-length clone encoding a putative G protein-coupled receptor. Homology analysis showed the highest similarity to M2 muscarinic acetylcholine receptors and overall low homology to all other biogenic amine receptors. Transfection of GPCR97 into a variety of cell lines conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine, but not to acetylcholine or any other biogenic amine. Subsequent analysis revealed a pharmacological profile practically indistinguishable from that for the histamine H3 receptor. In situ hybridization in rat brain revealed high levels of mRNA in all neuronal systems (such as the cerebral cortex, the thalamus, and the caudate nucleus) previously associated with H3 receptor function. Its widespread and abundant neuronal expression in the brain highlights the significance of histamine as a general neurotransmitter modulator. The availability of the human H3 receptor cDNA should greatly aid in the development of chemical and biological reagents, allowing a greater appreciation of the role of histamine in brain function.

Peptide-major histocompatibility complex protein complexes (pMHCs) on antigen-presenting cells (APCs) are central to T cell activation. Within minutes of peptide-specific T cells interacting with APCs, pMHCs on APCs formed clusters at the site of T cell contact. Thereafter, these clusters were acquired by T cells and internalized through T cell receptor-mediated endocytosis. During this process, T cells became sensitive to peptide-specific lysis by neighboring T cells (fratricide). This form of immunoregulation could explain the "exhaustion" of T cell responses that is induced by high viral loads and may serve to down-regulate immune responses.

Gene expression profiles of thousands of genes can now be examined en masse through cDNA and oligonucleotide microarrays 1-3 . Recently, studies have been reported that examined gene expression changes in yeast 4,5 , as well as in mammalian cell lines 6 , primary cells 7 and tissues 8 . However, present applications of microarray technology do not include the study of gene expression from individual cell types residing in a given tissue/organ (that is, in situ). Such studies would greatly facilitate our understanding of the complex interactions that exist in vivo between neighboring cell types in normal and disease states. We demonstrate here that gene expression profiles from adjacent cell types can be successfully obtained by integrating the technologies of laser capture microdissection 9 (LCM) and T7-based RNA amplification 10 with cDNA microarrays 11 . Neighboring small and large neurons are individually capturedTo demonstrate this integration of technologies, we examined the differential gene expression between large-and small-sized neurons in the dorsal root ganglia (DRG). In general, large DRG neurons are myelinated, fast-conducting and transmit mechanosensory information, whereas small neurons are unmyelinated, slow-conducting and transmit nociceptive information 12 . We chose this system because numerous differentially expressed genes (small versus large) have been reported, thus the success of this experiment could be assessed; and because many small and large neurons are adjacent to each other, thus we could test whether individual neurons can be cleanly captured. Large (diameter of >40 µm) and small (diameter <25 µm and with identified nuclei) neurons were cleanly and individually captured by LCM from sections (10 µm in thickness) of Nissl-stained rat DRG (Fig. 1). For this study, two sets of 1,000 large neurons and three sets of 1,000 small neurons were captured for cDNA microarray analysis. RNA amplification is reproducible between individual capturesRNA was extracted from each set of neurons and linearly amplified (independently) an estimated 10 6 -fold using T7 RNA polymerase. After being amplified, one fluorescently labeled probe was synthesized from an individually amplified RNA (aRNA), divided equally into three parts and hybridized in triplicate to a microarray ('chip') containing 477 cDNAs (see Methods for chip design) plus 30 cDNAs encoding plant genes (for the determination of non-specific nucleic acid hybridization). Expression in each neuronal set (called S1, S2 and S3 for small and L1 and L2 for large neurons) was thus monitored in triplicate, requiring a total of 15 microarrays. The quality of the microarray data is demonstrated by pseudocolor arrays, one resulting from hybridization to probes derived from neuronal set S1 and the other from neuronal set L1 (Fig. 2a). In Fig. 2a, the enlarged part of the chip shows some differences in fluorescence intensity (that is, expression levels) for particular cDNAs and demonstrates that spots containing the different cDNAs are relatively uniform ...

Each organelle of the secretory pathway is required to selectively allow transit of newly synthesized secretory and plasma membrane proteins and also to maintain a unique set of resident proteins that define its structural and functional properties. In the case of the endoplasmic reticulum (ER), residency is achieved in two ways: (a) prevention of residents from entering newly forming transport vesicles and (b) retrieval of those residents that escape. The latter mechanism is directed by discrete retrieval motifs: Soluble proteins have a H/KDEL sequence at their carboxy-terminus; membrane proteins have a dibasic motif, either di-lysine or di-arginine, located close to the terminus of their cytoplasmic domain. Recently it was found that di-lysine motifs bind the complex of cytosolic coat proteins, COP I, and that this interaction functions in the retrieval of proteins from the Golgi to the ER. Also discussed are the potential roles this interaction may have in vesicular trafficking.

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Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum. Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created. Incubation of soluble calnexin with a mixture of Glc0-3Man9GlcNAc2 oligosaccharides resulted in specific binding of the Glc1Man9GlcNAc2 species. Furthermore, Glc1Man5-7GlcNAc2 oligosaccharides bound relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues was important for binding. To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, alpha 1-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without disrupting the association with calnexin. Furthermore, the addition of 1 M alpha-methyl glucoside or alpha-methyl mannoside had no effect on complex stability. These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc1Man9GlcNAc2 oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.